The invention relates to new benzene derivatives containing at least two fluorine atoms, their use as NMR diagnostic agents, diagnostic agents that contain these benzene derivatives as well as a process for the production of these compounds and agents.
Modern medical technique makes it possible to represent the smallest morphological structures with a resolution that comes close to that of tissue sections from anatomy textbooks.
However, it is not possible even with the help of ultrasound, X-ray diagnosis, nuclear medicine and even nuclear spin tomography to obtain data on the metabolic physiological condition of a tissue of the living organism. But for a more accurate diagnosis and especially for planning and monitoring of a therapy this knowledge is of considerable importance since an optimal therapy can best be devised when early indications of its effects are possible.
An important parameter of the metabolic physiological activity is the pH. Many pathological processes result in a change of the hydrogen ion concentration. One of the best known examples is the release of lactic acid as a result of inadequate oxygen supply and the anaerobic metabolism of glucose it causes. Anaerobic glycolysis practically always occurs where a sufficient supply of oxygen is no longer guaranteed. A short-term acidification can be detected, for example, in areas of highest muscular activity. But here the accumulating lactic acid is carried away relatively quickly in the resting pause, so that no overacidifying can be detected in the resting muscle. But this appears to be different in areas of permanent oxygen debt. In ischemic areas (infarct) there is a shift of the pH because of the increased anaerobic glycolysis. Similar effects can be observed in rapidly growing neoplasms. Besides a disturbance of regulation, a lack of oxygen is present in the area of a tumor, so that here also an acidification occurs by anaerobic metabolism of carbohydrates.
The determination of the tissue pH thus leads to important statements on the function, condition and growth of cells, so that it is generally desirable to localize metabolic acidosis. (Am. J. Physiol. 246, R 409, 1984; R. Nuccitelli, D. W. Deamer, Eds. 1982 Intracellular pH: Its Measurement, Regulation and Utilization in Cellular Functions, Liss, New York.) Besides the measurement of the pH with pH electrodes, recently NMR spectroscopy has been used for this purpose. With its help, it was possible for the first time to determine the pH of the tissue noninvasively.
Determination of the pH with the help of NMR spectroscopy is based on the measurement of the signals of a chemical compound, which is in a pH-dependent, reversible equilibrium. If this equilibrium is slow relative to the NMR time scale, the signals of all components can be obtained and the signal intensities correspond to the concentrations of the equilibrium components. On the other hand, in the case of a rapid equilibrium, only one signal can be measured and the chemical shift is given by the chemical shift of the equilibrium components and their concentration.
In a two-component equilibrium, with the knowledge of the pK.sub.a and the chemical shift of the components, the pH can be calculated with the help of the Henderson-Hasselbalch equation.
It can be seen from the following table which atomic nuclei in principle are suitable for NMR imaging or spectroscopy:
__________________________________________________________________________ Relative Frequency Measurement Concentration Chemical at 1 tesia Sensitivity in biological Chemical Modification T.sub.1 Relaxation No. Nucleus MHz .sup.1 H = 1 tissue Shift Possibility times (seconds) __________________________________________________________________________ 1 .sup. 1 H 42.6 1.0 100 mol/l small very high 0.1-3 2 .sup.19 F 40.1 0.8 &lt;&lt;1 mmol/l very great very high 1-5 3 .sup.23 Na 11.3 0.09 100 mmol/l ./. practically zero &lt;0.1 4 .sup.31 P 17.2 0.06 10 mmol/l average limited 1-5 5 .sup.13 C 10.7 0.0002 1 mmol/l very great very high 1-10 __________________________________________________________________________
For 15 years the .sup.31 P nucleus has been used as a noninvasive measuring probe for intracellular pH measurement (J. Biol. Chem. 248, 7276, 1973). In this case the pH-sensitive signal is the signal of the inorganic phosphate from the hydrogen phosphate-dihydrogen phosphate equilibrium; the .sup.31 P signal of the phosphorus creatine serves as reference.
But the use of the .sup.31 P nucleus for pH determination also has its limits: thus an exact determination of the pH in a well-localized tissue volume in humans even with the use of 2 T nuclear spin tomographs is not possible. This is due to the relatively low phosphate concentrations as well as the fact that the .sup.31 P signal metrologically is difficult to register. Interfering signals in the area of the inorganic phosphate, superposition of the inorganic signal by other P metabolites or the lack of a reference signal can prevent a pH measurement. Other difficulties are in the slight sensitivity of the nucleus and the slight pH dependence of the chemical shift. The accuracy of the pH measurement is influenced above all by the determination of the chemical shifts of the signals and is no better than 0.2 pH units. Further, resonance signals can be entirely lacking in case of use of endogenous phosphates, since the compounds accumulate in such small concentrations (e.g., in intestines or in Ehrlich ascites tumor cells), that a pH determination is not possible.
Because of these circumstances only a quite inaccurate pH determination in comparatively large volumes is possible. Signals are picked up in an accumulation time of 15 minutes from a measuring volume of about 100 cc to provide a satisfactory .sup.31 P spectrum.
In case of use of a nucleus other than .sup.31 P the .sup.19 fluorine nucleus is the nucleus of choice, since it provides an easily measurable NMR signal, which is very similar to that of the hydrogen proton (it also has a nuclear spin of 1/2 like .sup.1 H), i.e., the same receiving and transmitting coils as in .sup.1 H NMR diagnosis can be used, it has a great sensitivity (about 83% of .sup.1 H), is available in 100% frequency and the signals are distributed over a great frequency range. Other advantages to be listed are the absence of fluorine in the organism (except for the teeth), so that no complications with endogenous F signals can occur (lack of a 19F background signal) as well as favorable chemical accessibility.
Data which can be obtained by the use of F molecules in NMR diagnosis cannot be obtained by any other diagnostic imaging or quasi-imaging process: The signal can greatly change in the body--depending on the chemical condition--thus allowing the quantification of biochemical reactions and making possible a direct observation of physiological processes. Despite these tempting properties, there is a problematic concentration consideration. A meaningful experiment requires .sup.19 F concentrations of greater than 1 mmol of F/1, i.e., the compounds to be applied must exhibit an outstanding tolerability and have a good solubility in water in order that in solutions of high concentration, the smallest possible volumes can be used.
The frequency (chemical shift) of a fluorine line is determined by the position of the F atom in the molecule. In principle, this also applies to all other atomic nuclei, but in the case of the fluorine atom the chemical shift is particularly strongly pronounced. A reference line is required to observe or quantify a shift of the fluorine signal. This frequency line can be the .sup.1 H signal, an external F standard or an unchanging F line, which is also located in the area to be measured. This reference line can be located in another, similarly distributing molecule or preferably in the molecule used as indicator. The most favorable situation is in the last-named case, since here only one substance is applied and no problems whatsoever with susceptibility effects occur so that an unequivocal assignment of the signals is possible.
Therefore, there is a need to find suitable compounds which react to a change of the pH with a changed measuring magnitude (resonance frequency) in the NMR spectrum with simultaneous presence of a reference line. Further, these compounds or diagnostic agents containing these compounds must exhibit the following properties:
(a) a great chemical shift per pH unit; PA1 (b) suitable pK values for in vivo measurements; PA1 (c) pharmacokinetics suitable for diagnosis; PA1 (d) a high accumulation in the target organs sufficient for a measurement; PA1 (e) good compatibility and low toxicity; PA1 (f) metabolic stability; PA1 (g) great chemical stability and shelf life; and PA1 (h) good water solubility.
The compounds described so far (and only for in vitro examinations) (Annals of the New York Academy of Science, S. M. Cohen, Ed. 1987, 508, 33) do not meet these requirements. Thus, with them, e.g., a pH determination more accurate than with .sup.31 P is not possible, since the pH dependence of the chemical shift is too small (smaller than or equal to 1 ppm/pH) and/or their pH values are outside the physiological range and/or their resonance frequencies are dependent not only on the pH but also on the field strength. Also the described compounds, because of their poor compatibility, are not suitable for an animal experiment or not at all for clinical use.